Table 1.

Detailed protocol for cell-based TMPRSS2 enzyme inhibitory assay using VeroE6/TMPRSS2 cells.

Step no.	Process	Notes
1	Cells plated at a density of 10,000 viable cells/well in triplicate in 96-well microplate with 100 μL/well culture medium.	DMEM (e.g., Corning cat.# 10-013-CV), supplemented with 10% heat inactivated fetal bovine serum (e.g., Millipore-Sigma cat.# 12306C-100 ML), 100 unit/mL penicillin and streptomycin, and 1.0 mg/mL geneticin used as culture medium. Black and clear bottom 96-well plate (e.g., Corning Corsta 3603).
2	The plate incubated at 37 °C and 5% CO2 for 24 h
3	Take out the plate from the incubator, remove the culture medium completely. Wash wells with 100 μL/well of DPBS (1x) and completely remove the DPBS.	The monolayer of cells in the wells should stay at the bottom of the wells when washing. The DPBS (1x) buffer should be without calcium, magnesium, and phenol red (e.g., Corning cat# 21-031-cv)
4	Add 50 μL/well of inhibitor solution in DPBS (1x) and stand at RT for 5 min.	The inhibitor stock solution prepared by dissolving the inhibitor in DMSO or H2O. Stock solutions diluted with DPBS (1x). Final DMSO concentration should be less than 0.2%.
5	Add 50 μL/well of substrate solution in DPBS (1x).
6	Read fluorescence intensity after 30 min at excitation 360 nm/emission 460 nm.	Instrument parameter settings is depend on the microplate reader used. For LJL Biosystems Analyst AD, a default parameters, including Z height: middle of the well; integration time: 100 ms; Attenuator: off; PMT mode: SmartRead with sensitivity 2; Plate shaking time: 10 s; Optics: bottom with 50/50 dichroic mirror.